Lymphotactin (Ltn) is the prototype and single member of the C class of chemokines. This small secreted protein signals T lymphocytes and NK cells by specific activation of the XCR1 G protein-coupled receptor protein, drawing them to the site of an injury or infection. Demonstrated roles in tumor regression and tissue transplant rejection highlight two disease states that may respond to treatment with either Ltn mimetics or antagonists. The Ltn amino acid sequence distinguishes it from related proteins through its lack of one of the two disulfide bonds found in all the other chemokines, as well as a unique C-terminal extension of approximately 25 residues. Our structural investigations of Ltn by NMR spectroscopy showed that the protein exists in two distinct conformational states, one of which coincides with the conserved structural arrangement seen in all other chemokines. In Specific Aim I, we will test the hypothesis that only one of these Ltn conformers binds and signals through the XCR1 receptor. Immunoprecipitation and fluorescence approaches will be developed for detection of Ltn binding to the XCR1 receptor, while receptor signaling by Ltn will be assayed from inhibition of intracellular cyclic AMP levels. These studies will reveal which of the two Ltn structures is biologically active. In addition to signaling through its high-affinity interactions with XCR1, Ltn binds cell surface glycosaminoglycans (GAGs), as do most chemokines. Experiments in Aim II will map the GAG binding surface of Ltn through mutagenesis, surface plasmon resonance binding measurements and NMR. XCR1 binding and signaling will be evaluated for soluble Ltn:GAG complexes and for Ltn mutants that fail to bind GAGs in order to test the hypothesis that GAG binding influences receptor recognition. Experiments in Specific Aim III will determine the three-dimensional structure of the alternative conformation of Ltn and develop a structure to activity algorithm to construct Ltn mutants with constrained 3D structures. Further goals of Specific Aim III are the identification of specific residues in the Ltn C-terminal extension that are required for its activity, as well as other structural elements that participate in receptor binding and activation.